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Dehydroepiandrosterone (DHEA) has a promising market due to its capacity to regulate human hormone levels as well as preventing and treating various diseases. We have established a chemical esterification coupled biocatalytic-based scheme by lipase-catalyzed 4-androstene-3,17-dione (4-AD) hydrolysis to obtain the intermediate product 5-androstene-3,17-dione (5-AD), which was then asymmetrically reduced by a ketoreductase from Sphingomonas wittichii (SwiKR). Co-enzyme required for KR is regenerated by a glucose dehydrogenase (GDH) from Bacillus subtilis. This scheme is more environmentally friendly and more efficient than the current DHEA synthesis pathway. However, a significant amount of 4-AD as by-product was detected during the catalytic process. Focused on the control of by-products, we investigated the source of 4-AD and identified that it is mainly derived from the isomerization activity of SwiKR and GDH. Increasing the proportion of glucose in the catalytic system as well as optimizing the catalytic conditions drastically reduced 4-AD from 24.7 to 6.5% of total substrate amount, and the final yield of DHEA achieved 40.1 g/L. Furthermore, this is the first time that both SwiKR and GDH have been proved to be promiscuous enzymes with dehydrogenase and ketosteroid isomerase (KSI) activities, expanding knowledge of the substrate diversity of the short-chain dehydrogenase family enzymes. Key points: • A strategy of coupling lipase, ketoreductase, and glucose dehydrogenase in producing DHEA from 4-AD • Both SwiKR and GDH are identified with ketosteroid isomerase activity. • Development of catalytic strategy to control by-product and achieve highly selective DHEA production [ABSTRACT FROM AUTHOR]

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-β-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/β hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/β hydrolase core composed of a parallel, six-stranded β-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/β hydrolases based on our results. Key points: • AtABH is a promiscuous αβ hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αβ hydrolases are a possible source of new OTA-degrading enzymes [ABSTRACT FROM AUTHOR]

Bibenzyls, a kind of important plant polyphenols, have attracted growing attention for their broad and remarkable pharmacological activities. However, due to the low abundance in nature, uncontrollable and environmentally unfriendly chemical synthesis processes, these compounds are not readily accessible. Herein, one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes. Three types of efficiently post-modifying modular strains were engineered by employing methyltransferases, prenyltransferase, and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules. Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes. Especially, a prenylated bibenzyl derivative (12) was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke. RNA-seq, quantitative RT-PCR, and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor, mitochondria associated 3 (Aifm3), suggesting that Aifm3 might be a new target in ischemic stroke therapy. This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery.

Bibenzyls, a kind of important plant polyphenols, have attracted growing attention for their broad and remarkable pharmacological activities. However, due to the low abundance in nature, uncontrollable and environmentally unfriendly chemical synthesis processes, these compounds are not readily accessible. Herein, one high-yield bibenzyl backbone-producing Escherichia coli strain was constructed by using a highly active and substrate-promiscuous bibenzyl synthase identified from Dendrobium officinale in combination with starter and extender biosynthetic enzymes. Three types of efficiently post-modifying modular strains were engineered by employing methyltransferases, prenyltransferase, and glycosyltransferase with high activity and substrate tolerance together with their corresponding donor biosynthetic modules. Structurally different bibenzyl derivatives were tandemly and/or divergently synthesized by co-culture engineering in various combination modes. Especially, a prenylated bibenzyl derivative (12) was found to be an antioxidant that exhibited potent neuroprotective activity in the cellular and rat models of ischemia stroke. RNA-seq, quantitative RT-PCR, and Western-blot analysis demonstrated that 12 could up-regulate the expression level of an apoptosis-inducing factor, mitochondria associated 3 (Aifm3), suggesting that Aifm3 might be a new target in ischemic stroke therapy. This study provides a flexible plug-and-play strategy for the easy-to-implement synthesis of structurally diverse bibenzyls through a modular co-culture engineering pipeline for drug discovery. Structurally diverse bibenzyl derivatives were tandemly and divergently synthesized by modular co-culture engineering, and a potent neuroprotective agent was discovered after in vitro and in vivo activity evaluation. [Display omitted] [ABSTRACT FROM AUTHOR]

Terpenoids are ubiquitous to all kingdoms of life and are one of the most diverse groups of compounds, both structurally and functionally. Despite being derived from common precursors, isopentenyl diphosphate and dimethylallyl diphosphate, their exceptional diversity is partly driven by the substrate and product promiscuity of terpene synthases that produce a wide array of terpene skeletons. Plant terpene synthases can be subdivided into different subfamilies based on sequence homology and function. However, in many cases, structural architecture of the enzyme is more essential to product specificity than primary sequence alone, and distantly related terpene synthases can often mediate similar reactions. As such, the focus of this brief review is on some of the recent progress in understanding terpene synthase function and diversity. [ABSTRACT FROM AUTHOR]

Harnessing enzymes which possess several catalytic activities is a topic where intense research has been carried out, mainly coupled with the development of cascade reactions. This review tries to cover the different possibilities to reach this goal: enzymes with promiscuous activities, fusion enzymes, enzymes + metal catalysts (including metal nanoparticles or site-directed attached organometallic catalyst), enzymes bearing non-canonical amino acids + metal catalysts, design of enzymes bearing a second biological but artificial active center (plurizymes) by coupling enzyme modelling and directed mutagenesis and plurizymes that have been site directed modified in both or in just one active center with an irreversible inhibitor attached to an organometallic catalyst. Some examples of cascade reactions catalyzed by the enzymes bearing several catalytic activities are also described. Finally, some foreseen problems of the use of these multi-activity enzymes are described (mainly related to the balance of the catalytic activities, necessary in many instances, or the different operational stabilities of the different catalytic activities). The design of new multi-activity enzymes (e.g., plurizymes or modified plurizymes) seems to be a topic with unarguable interest, as this may link biological and non-biological activities to establish new combo-catalysis routes. [ABSTRACT FROM AUTHOR]

The ability to catalyse a reaction acting on different substrates, known as “broad-specificity” or “multi-specificity”, and to catalyse different reactions at the same active site (“promiscuity”) are common features among the enzymes. These properties appear to go against the concept of extreme specificity of the catalytic action of enzymes and have been re-evaluated in terms of evolution and metabolic adaptation. This paper examines the potential usefulness of a differential inhibitory action in the study of the susceptibility to inhibition of multi-specific or promiscuous enzymes acting on different substrates. Aldose reductase is a multi-specific enzyme that catalyses the reduction of both aldoses and hydrophobic cytotoxic aldehydes and is used here as a concrete case to deal with the differential inhibition approach.

The promiscuous activities of enzymes provide fertile ground for the evolution of new metabolic pathways. Here, we systematically explore the ability of E. coli to harness underground metabolism to compensate for the deletion of an essential biosynthetic pathway. By deleting all threonine deaminases, we generated a strain in which isoleucine biosynthesis was interrupted at the level of 2-ketobutyrate. Incubation of this strain under aerobic conditions resulted in the emergence of a novel 2-ketobutyrate biosynthesis pathway based upon the promiscuous cleavage of O-succinyl-L-homoserine by cystathionine γ-synthase (MetB). Under anaerobic conditions, pyruvate formate-lyase enabled 2-ketobutyrate biosynthesis from propionyl-CoA and formate. Surprisingly, we found this anaerobic route to provide a substantial fraction of isoleucine in a wild-type strain when propionate is available in the medium. This study demonstrates the selective advantage underground metabolism offers, providing metabolic redundancy and flexibility which allow for the best use of environmental carbon sources.

The ability to catalyse a reaction acting on different substrates, known as "broad-specificity" or "multi-specificity", and to catalyse different reactions at the same active site ("promiscuity") are common features among the enzymes. These properties appear to go against the concept of extreme specificity of the catalytic action of enzymes and have been re-evaluated in terms of evolution and metabolic adaptation. This paper examines the potential usefulness of a differential inhibitory action in the study of the susceptibility to inhibition of multi-specific or promiscuous enzymes acting on different substrates. Aldose reductase is a multi-specific enzyme that catalyses the reduction of both aldoses and hydrophobic cytotoxic aldehydes and is used here as a concrete case to deal with the differential inhibition approach. [ABSTRACT FROM AUTHOR]

Engineering biotechnological microorganisms to use methanol as a feedstock for bioproduction is a major goal for the synthetic metabolism community. Here, we aim to redesign the natural serine cycle for implementation in E. coli. We propose the homoserine cycle, relying on two promiscuous formaldehyde aldolase reactions, as a superior pathway design. The homoserine cycle is expected to outperform the serine cycle and its variants with respect to biomass yield, thermodynamic favorability, and integration with host endogenous metabolism. Even as compared to the RuMP cycle, the most efficient naturally occurring methanol assimilation route, the homoserine cycle is expected to support higher yields of a wide array of products. We test the in vivo feasibility of the homoserine cycle by constructing several E. coli gene deletion strains whose growth is coupled to the activity of different pathway segments. Using this approach, we demonstrate that all required promiscuous enzymes are active enough to enable growth of the auxotrophic strains. Our findings thus identify a novel metabolic solution that opens the way to an optimized methylotrophic platform. Image 1 • We designed the homoserine cycle as an efficient route for methylotrophic growth. • The homoserine cycle can outcompete all natural methanol assimilation routes. • The homoserine cycle is based on three enzyme promiscuous activities. • We selected for the in vivo activity of serine aldolase as a source of serine. • We selected for the activity of HOB aldolase and aminase as source of amino acids. [ABSTRACT FROM AUTHOR]

The metabolic pathways that carry out the biochemical transformations sustaining life depend on the efficiency of their associated enzymes. In recent years, it has become clear that promiscuous enzymes have played an important role in the function and evolution of metabolism. In this work we analyze the repertoire of promiscuous enzymes in 89 non-redundant genomes of the Archaea cellular domain. Promiscuous enzymes are defined as those proteins with two or more different Enzyme Commission (E.C.) numbers, according the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. From this analysis, it was found that the fraction of promiscuous enzymes is lower in Archaea than in Bacteria. A greater diversity of superfamily domains is associated with promiscuous enzymes compared to specialized enzymes, both in Archaea and Bacteria, and there is an enrichment of substrate promiscuity rather than catalytic promiscuity in the archaeal enzymes. Finally, the presence of promiscuous enzymes in the metabolic pathways was found to be heterogeneously distributed at the domain level and in the phyla that make up the Archaea. These analyses increase our understanding of promiscuous enzymes and provide additional clues to the evolution of metabolism in Archaea. [ABSTRACT FROM AUTHOR]

Background Leishmania infantum is a protozoan of the trypanosomatid family causing visceral leishmaniasis. Leishmania parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS 2 ), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival. Methods The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements. Results The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the K M for NADPH decreases six times compared to the value obtained by adding TS 2 as last reagent to start the reaction (1.9 vs. 12 μM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O 2 to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O 2 oxidoreductase activity is shared by Saccharomyces cerevisiae glutathione reductase (GR). Conclusions TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water. General significance TR and GR are promiscuous enzymes. [ABSTRACT FROM AUTHOR]

The mercury resistance pathway enzyme organomercurial lyase (MerB) catalyzes the conversion of organomercurials to ionic mercury (Hg 2+ ). Here, we provide evidence for the emergence of this enzyme from a TRASH-like, non-enzymatic, treble-clef zinc finger ancestor by domain duplication and fusion. Surprisingly, the structure-stabilizing metal-binding core of the treble-clef appears to have been repurposed in evolution to serve a catalytic role. Novel enzymatic functions are believed to have evolved from ancestral generalist catalytic scaffolds or from already specialized enzymes with catalytic promiscuity. The emergence of MerB from a zinc finger ancestor serves as a rare example of how a novel enzyme may emerge from a non-catalytic scaffold with a related binding function. [ABSTRACT FROM AUTHOR]

Enzymes are an attractive alternative in the asymmetric syntheses of chiral building blocks. To meet the requirements of industrial biotechnology and to introduce new functionalities, the enzymes need to be optimized by protein engineering. This article specifically reviews rational approaches for enzyme engineering and de novo enzyme design involving structure-based approaches developed in recent years for improvement of the enzymes’ performance, broadened substrate range, and creation of novel functionalities to obtain products with high added value for industrial applications.

Protein tyrosine phosphatases (PTPs) fulfil multiple key regulatory functions. Within the group of PTPs, the atypical lipid phosphatases (ALPs) are known for their role as virulence factors associated with human pathogens. Another group of PTPs, which is capable of using inositol-hexakisphosphate (InsP6) as substrate, are known as phytases. Phytases play major roles in the environmental phosphorus cycle, biotechnology, and pathogenesis. So far, all functionally characterized PTPs, including ALPs and PTP-phytases, have been derived exclusively from isolated microorganisms. In this study, screening of a soil-derived metagenomic library resulted in identification of a gene (pho16B), encoding a PTP, which shares structural characteristics with the ALPs. In addition, the characterization of the gene product (Pho16B) revealed the capability of the protein to use InsP6 as substrate, and the potential of soil as a source of phytases with so far unknown characteristics. Thus, Pho16B represents the first functional environmentally derived PTP-phytase. The enzyme has a molecular mass of 38 kDa. The enzyme is promiscuous, showing highest activity and affinity toward naphthyl phosphate (Km 0.966 mM). Pho16B contains the HCXXGKDR[TA]G submotif of PTP-ALPs, and it is structurally related to PtpB of Mycobacterium tuberculosis. This study demonstrates the presence and functionality of an environmental gene codifying a PTP-phytase homologous to enzymes closely associated to bacterial pathogenicity. [ABSTRACT FROM AUTHOR]